Regulation of connexin 43 and microRNA expression via β2-adrenoceptor signaling in 1321N1 astrocytoma cells

Abstract

Abstract. Connexin 43 (Cx43) is the main gap junction protein in astrocytes and exerts the same effects on growth inhibition in astrocytoma and glioma as microRNA-146a (miR-146a) in glioma. β2-adrenergic receptor (AR) signaling modulates Cx43 expression in myocytes via components downstream of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). However, it remains to be elucidated how expression of Cx43 is modulated in astrocytes. In the present study, 1321N1 astrocytoma cells were treated with β2-AR signaling agents in order to evaluate the expression of Cx43 and miRNAs. RNA and protein were extracted from the cells for use in reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The results revealed that clenbuterol increased miR-146a level and upregulated Cx43 expression via cAMP/PKA at the mRNA and protein level. Pre-inhibition of adenyl cyclase decreased expression of Cx43 and miR-146a. PKA activation and overexpression of miR-146a in A-1321N1 cells increased the expression of Cx43. β2-AR stimulation and 6Bnz, a PKA activator, suppressed oncomiRs miR-155 and miR-27a, while 8-(4-chlorophenylthio)-2'-O-methyl adenosine-3',5'-cyclic monophosphate, an Epac activator, increased their levels. The current findings demonstrated that β2-AR signaling has growth inhibitory effects via modulation of the cAMP/PKA pathway in A-1321N1 cells through increasing the expression level of Cx43 and miR-146a as well as decreasing miR-155 and miR-27a levels. Thus, stimulation of the β2-AR and PKA signaling pathway may be a useful approach for astrocytoma therapy.

β2-Adrenergic receptor agonist enhances the bystander effect of HSV-TK/GCV gene therapy in glioblastoma multiforme via upregulation of connexin 43 expression

“… 35 Consistent with our results, other studies have shown that the signaling pathways induced by β2-AR stimulation lead to the upregulation of Cx43 levels in various cells. 33 , 34 , 44 , 45 , 46 Since Cx43 is the major structure of GJIC, which is an important factor in GCV-TP transmission, we evaluated the effect of Cln on apoptosis induction of HSV-TK/GCV gene therapy in GBM cells. The question was addressed as to whether increased Cx43 expression leads to the induction of apoptosis by HSV-TK/GCV in more cells and thus increases the efficiency of gene therapy.…”

“… 25 , 27 , 31 , 32 β2-Adrenergic receptor (β2-AR) signaling is an important pathway to the regulation of Cx43 levels. 33 , 34 Our previous study indicated that clenbuterol hydrochloride (Cln), a selective β2-AR agonist, enhanced the Cx43 expression levels in human GBM cells and OECs. 35 In this study, we hypothesize that the stimulation of β2-AR via the upregulation of Cx43 expression strengthens bystander effect and improves the gene therapy in GBM cells ultimately.…”

Epac: A Promising Therapeutic Target for Vascular Diseases: A Review

“…Surgery is difficult because of the migratory and invasive characteristics of glioma cells and had no discernible effect on survival. Connexin 43 (Cx43), a key gap junction-forming protein, has been found to be inversely related to the degree of malignancy in glioma ( Mostafavi et al, 2014 ; Khaksarian et al, 2015 ). A previous study showed that clenbuterol hydrochloride can increase Cx43 and miR-451 to suppress human astrocytoma cells via the cAMP/Epac ( Mostafavi et al, 2014 ).…”

“…Epac signaling promotes the expression of Cx43 and miR-451. However, another study reported that Epac activation can increase the levels of oncomiRs, miR-155, and miR-27a while decreasing the levels of miR-146a ( Khaksarian et al, 2015 ). Stimulation of the Epac pathway promotes astrocytoma development.…”

The Astrocytic cAMP Pathway in Health and Disease

“…cAMP can regulate connexin expression and gap junction assembly. A β-adrenergic agonist has been shown to upregulate the expression of Cx43 in glioma cell lines by activating Epac [84,85]. Corticotropin-releasing hormones can increase Cx43 expression and gap junction communication in cultured astrocytes and organotypic hippocampal slice cultures via the PKA pathway [86].…”